Study On Oxidative Stress,Mitochondrial Damage And Pyroptosis Of Mice Liver Induced By Ageratina Adenophora

Ageratina adenophora（A.adenophora）is an invasive weed that spread widely distributed in many parts of the world.It poses a serious threat to the agriculture,forestry and animal husbandry industry.Ingestion of A.adenophora by animals leads to poisoning conditions and sometimes death.Current researches have reported the liver as an important target organ for A.adenophora toxicity.However,the scientific basis for its toxicological effects is finite,the inflammatory responses caused by A.adenophora is poorly elucidated.In this study,80 eight-week-old Kunming mice were randomly divided into four groups:control group and A.adenophora administration groups（100 g/kg,200 g/kg and 300 g/kg）.The purpose of this study was to expound the possible mechanism of hepatotoxicity induced by A.adenophora from the perspectives of oxidative stress,mitochondrial（Mt）damage,and pytoptosis at the tissue,cellular and molecular levels.Compared with the control groups,the main results were as follows:1.Inflammatory damage induced by A.adenophora in mice liverAfter clinical observation,histopathology and molecular biology research,all A.Adenophora administration groups showed symptoms like loss of appetite,weight loss,mental disorder,rough hair and other toxic symptoms.Compared with the control group,the levels of alkaline phosphatase（ALP）,alanine aminotransferase（ALT）and aspartate aminotransferase（AST）of serum in the A.adenophora administrated groups were all significantly increased in a dose-dependent manner（p<0.01）.In addition,A.adenophora signigicantly elevated the hepatosomatic indexes（HSI）when compared to the control group（p<0.01）.Furthermore,the histopathological changes that were observed during the liver pathological examination included hepatocyte swelling,hemorrhage,disorder of arrangement of hepatic cord,inflammatory cell infiltration,hyperplasia of nodal hoof tissue and other pathological changes.However,there was no obvious pathological damage in the other major parenchymal organs.The mRNA expressions of TNF-αand IL-6 in the A.adenophora group were significantly increased（p<0.01）as compared to the control group,while the IL-4 and IL-10 mRNA expression levels in A.adenophora groups were remarkably decreased in a dose-dependent manner（p<0.01）.These results indicated that,≥100 g/kg dose of A.Adenophora could induce inflammatory damage to the liver of mice.2.A.adenophora unbalanced the oxidation system and caused oxidative stress in liverIn this study,the effect of A.Adenophora on oxidative stress system of mice liver was researched by using Flow cytometry（FCM）,chemical analysis and quantitative real time PCR（qRT-PCR）.The results showed that:（1）A.Adenophora could significantly promote the production of ROS in hepatocytes and also increased MDA production in liver tissue among treated groups in a dose-dependent manner（p<0.01）;（2）A.Adenophora could significantly reduced the activities of CAT（p<0.01）,GSH Px（p<0.01）,Cu-Zn SOD and Mn-SOD in the liver of mice by comparing with the control group.Also,A.Adenophora significantly decrease the level of GSH among all treated groups in a dose-dependent manner（p<0.01）;（3）Furthermore,A.adenophora decreased the mRNA expression levels of CAT,GSH-Px,Mn-SOD,Cu-Zn SOD of liver in a dose-dependent manner（p<0.05 or p<0.01）.The results above showed that,≥100 g/kg dose of A.Adenophora damaged the redox system and caused oxidative stress in the liver of mice.3.A.adenophora caused the mitochondrial damage in miceIn this study,FCM,transmission electron microscopy（TEM）and qRT-PCR methods were used to investigate the effect of A.Adenophora on the structure and function of liver Mitochondrion（Mt）.The results showed（1）crista abnormalities in the 100 g/kg group,however,these crista abnormalities were heterogeneous in size and shapes in the 200 g/kg treated group.In addition,partially swollen mitochondria with unusual and sparse cristae were observed in300 g/kg A.adenophora-treated groups;（2）Furthermore,we determined the quantitative degree of mitochondrial swelling using FCM.It showed that the size of Mt in all A.adenophora treated groups were remarkablely larger than that in the control group（p<0.01）;（3）In addition,to detect the effect of A.adenophora on the mitochondrial function,ATPase activities(Na⁺K⁺-ATPase and Ca²⁺Mg²⁺-ATPase)were measured in the control and A.adenophora administration groups.The results showed that A.adenophora treatments led to decrease in the contents of Na⁺K⁺-ATPase and Ca²⁺Mg²⁺-ATPase in a dose-dependent manner when compared with the control group（p<0.01）;（4）In an effort to understand the changes in the levels of mtDNA,the mtDNA copy number was measured through qRT-PCR method.The copy number of mtDNA showed a decreasing trend in the A.adenophora-treated groups when compared to the control group（p<0.01）.Theses results showed that,≥100 g/kg dose of A.Adenophora could destroy the ultrastructure of Mt and lead to mitochondrial dysfunction.4.A.adenophora induced mice hepatotoxicity via ROS-NLRP3-mediated pyroptosisThe purpose of this study was to illuminate the effects of A.adenophora on pyroptosis signal pathway in the murine liver through FCM,qRT-PCR and Western blot.The results showed that:（1）A.adenophora significantly decreased the percentage of normal hepatocytes when compared with the control（p<0.01）.Whereas the percentage of PI positive pyroptosis hepatocytes in A.adenophora administrated groups were remarkably increased in a dose dependent manner（p<0.01）;（2）Compared with the control,the IL-βlevels in both serum and liver homogenate were all elevated in A.adenophora treated group（p<0.01）;（3）Moreover,caspase-1 activity was also augmented significantly in the A.adenophora administration groups（p<0.01）;（4）Two key pyroptosis factors were assayed by immunohistochemistry method.It showed that the optical density value of Caspase-1 and IL-βin liver were both increased in a dose-dependent manner（p<0.01）;（4）Gasdermin-D（GSDMD）,the downstream factor of Caspase-1,is a key executioner in pyroptosis pathway.qRT-PCR demonstrated that A.adenophora decreased the mRNA expression levels of GSDMD as well as other pytoptosis related gene such as NLRP3,NF-κB,Caspase-1,GSDMD and IL-1β（p<0.05 or p<0.01）;（5）The formation N-terminal of GSDMD（GSDMD-N）,is a cleavage body of GSDMD that promotes membrane rupture leading to IL-1βrelease.Western blot results showed that GSDMD-N occurred only in A.adenophora-treated groups,but not in the control group.This data showed that pyroptosis was induced by A.adenophora through Caspase-1 dependent pathway.To conclude,our study demonstrated that,A.adenophora caused liver inflammatory damage by inducing oxidative stress which produced a large number of ROS and then promoted the expression of inflammatory factors,causing structural and functional disorder of Mt and activating the signal pathway of pyroptosis,and finally caused the inflammatory damage of liver.This study provides a scientific basis for underlying the mechanisms of hepatotoxicity induced by A.adenophora in mice.